The present invention relates generally to cytokine receptors, and more specifically, to Interleukin-1 receptors.
Interleukin-1.alpha. and Interleukin-1.beta. (IL-1.alpha. and IL-1.beta.) are distantly related polypeptide hormones which play a central role in the regulation of immune and inflammatory responses. These two proteins were originally both classified as IL-1, based on a shared lymphocyte activation factor (LAF) activity, and a common major cellular source, activated macrophages. As information has accumulated from studies using purified natural and recombinant IL-1 molecules, it has become clear that IL-1.alpha. and IL-1.beta. each mediate most, if not all, of the wide range of activities previously ascribed to IL-1. The basis for this nearly identical spectrum of biological activities is thought to be a single class of plasma membrane IL-1 receptors which bind both IL-1.alpha. and IL-1.beta..
A few preliminary reports concerning the existence of an IL-1 plasma membrane receptor have been published. To date, structural characterization of the Interleukin-1 receptor has been limited to estimates of the molecular weight of this protein by gel filtration, by SDS-PAGE analysis of covalent complexes formed by chemical crosslinking between the receptor and .sup.125 I-IL-1 molecules, and by immunoprecipitation of labeled surface proteins.
Dower et al. (J. Exp. Med. 162:501, 1985), and Dower et al. (Proc. Natl. Acad. Sci. USA 83:1060, 1986), describe chemical crosslinking studies indicating an apparent 79.5 kilodalton (kDa) plasma membrane protein on LBRM-33-1A5 murine T lymphoma cells and a 78 kDa surface protein on a murine fibroblast cell line which bound .sup.125 I-labeled human Interleukin-1.beta.. Kilian et al. (J. Immunol. 136:4509, 1986) reported that murine .sup.125 I-IL-1.alpha. binding to murine thymoma cells could be blocked by human IL-1.alpha. and IL-1.beta.. Dower et al. (Nature 324:266, 1986) reported binding competition studies indicating that IL-1.alpha. and IL-1.beta. bound to the same cell surface receptors on LBRM-33-1A5 cells, human dermal fibroblasts, murine BALB-3T3 cells, and ARH77, a human B lymphoblastoid cell line. The receptors in the different cell lineages exhibited similar but not identical binding characteristics. The IL-1 receptors on porcine synovial fibroblasts (Bird et al., Nature 324:263, 1986) and human dermal fibroblasts (Chin et al., J. Exp. Med. 165:70, 1987) have been shown to yield a major species in the size range M.sub.r 97,000-100,000 when crosslinked to labeled IL-1, suggesting that a protein of M.sub.r 80,000 was responsible for binding IL-1. In contrast, IL-1 receptors characterized in this fashion on human B cells (Matsushima et al., J. Immunol. 136:4496, 1986) displayed an apparent molecular weight of 60,000.
Bron and MacDonald, FEBS Letters 219:365 (1987), disclose immunoprecipitation of murine IL-1 receptor from surface-labeled EL-4 cells using a rabbit polyclonal antiserum directed to IL-1. This work indicated that the murine receptor is a glycoprotein having an apparent molecular weight of approximately 82,000 daltons.
Radiolabeled IL-1 has been used in chemical crosslinking studies and for the detection of receptor in detergent extracts of cells. The results of these experiments, noted above, suggest that a protein of M.sub.r 60,000 or 80,000 is responsible for binding IL-1. The crosslinking of radiolabeled IL-1 to cells has also led to the occasional detection of proteins distinct from the major species of M.sub.r 80,000, suggesting that the IL-1 binding molecule may exist in the membrane as part of a multi-subunit receptor complex.
In order to study the structure and biological characteristics of IL-1 receptors and the role played by IL-1 receptors in the responses of various cell populations to IL-1 stimulation, or to use IL-1 receptors effectively in therapy, diagnosis, or assay, homogeneous compositions of IL-1 receptor are needed. Such compositions are theoretically available via purification of solubilized receptors expressed by cultured cells, or by cloning and expression of genes encoding the receptors. However, prior to the present invention, several obstacles prevented these goals from being achieved.
Even in cell lines known to express detectable levels of IL-1 receptor, the IL-1 receptor is present as a very minor component of total cellular proteins. Moreover, no cell lines were known that expressed high levels of IL-1 receptors constitutively and continuously. For example, the murine EL-4 6.1 cell line expresses detectable levels of IL-1 receptor, but the level of IL-1 receptor expression tends to decay with time, which greatly complicates the process of obtaining sufficient quantities of receptor to provide a useful starting material for purification. Thus, a method of continuously selecting cells for acceptable levels of IL-1 receptor expression, employing fluorescence-activated cell sorting (FACS), was devised.
Additional problems are inherent in attempting to clone mammalian genes encoding IL-1 receptor. Even if a protein composition of sufficient purity can be obtained to permit N-terminal protein sequencing, the degeneracy of the genetic code typically does not permit one to define a suitable probe without considerable additional experimentation. Many iterative attempts may be required to define a probe having the requisite specificity to identify a hydridizing sequence in a cDNA library. To circumvent this problem, a novel direct receptor expression cloning technique was devised to avoid the need for repetitive screening using different probes of unknown specificity. This technique, which has never before been employed, allows direct visualization of receptor expression following transfection of a mammalian cell line with a high expression vector containing a cDNA clone encoding the receptor.
Purified IL-1 receptor compositions will be useful in diagnostic assays for IL-1 or IL-1 receptor, and also in raising antibodies to IL-1 receptor for use in diagnosis or therapy. In addition, purified IL-1 receptor compositions may be used directly in therapy to bind or scavenge IL-1, thereby providing a means for regulating the immune or inflammatory activities of this cytokine.